Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root

Cytokinin (CK) influences many aspects of plant growth and development, and its function often involves intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood. Here we describe the isolation and characterization of the Arabidopsis CK-induced root curling 1 (ckrc1) mutant. CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. The ckrc1 mutant exhibits a defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin or IPA. Furthermore, we show that CK up-regulates CKRC1/TAA1 expression but inhibits polar auxin transport in roots in an AHK3/ARR1/12-dependent and ethylene-independent manner. Our results suggest that CK regulates root growth and development not only by down-regulating polar auxin transport, but also by stimulating local auxin biosynthesis.     

TAA1-Regulated Local Auxin Biosynthesis in the Root-Apex Transition Zone Mediates the Aluminum-Induced Inhibition of Root Growth in Arabidopsis

The transition zone (TZ) of the root apex is the perception site of Al toxicity. Here, we show that exposure of Arabidopsis thaliana roots to Al induces a localized enhancement of auxin signaling in the root-apex TZ that is dependent on TAA1, which encodes a Trp aminotransferase and regulates auxin biosynthesis. TAA1 is specifically upregulated in the root-apex TZ in response to Al treatment, thus mediating local auxin biosynthesis and inhibition of root growth. The TAA1-regulated local auxin biosynthesis in the root-apex TZ in response to Al stress is dependent on ethylene, as revealed by manipulating ethylene homeostasis via the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid, the inhibitor of ethylene biosynthesis aminoethoxyvinylglycine, or mutant analysis. In response to Al stress, ethylene signaling locally upregulates TAA1 expression and thus auxin responses in the TZ and results in auxin-regulated root growth inhibition through a number of auxin response factors (ARFs). In particular, ARF10 and ARF16 are important in the regulation of cell wall modification–related genes. Our study suggests a mechanism underlying how environmental cues affect root growth plasticity through influencing local auxin biosynthesis and signaling.     

Disturbed Local Auxin Homeostasis Enhances Cellular Anisotropy and Reveals Alternative Wiring of Auxin-ethylene Crosstalk in Brachypodium distachyon Seminal Roots

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2l^hypo). Counterintuitive however, Bdtar2l^hypo mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2l^hypo root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.     

Multilevel interactions between ethylene and auxin in Arabidopsis roots

Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants.     

A Link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis

The plant hormone ethylene participates in the regulation of a variety of developmental processes and serves as a key mediator of plant responses to biotic and abiotic stress factors. The diversity of ethylene functions is achieved, at least in part, by combinatorial interactions with other hormonal signals. Here, we show that ethylene-triggered inhibition of root growth, one of the classical effects of ethylene in Arabidopsis thaliana seedlings, is mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1) genes that encode alpha- and beta-subunits of a rate-limiting enzyme of Trp biosynthesis, anthranilate synthase. Upregulation of WEI2/ASA1 and WEI7/ASB1 by ethylene results in the accumulation of auxin in the tip of primary root, whereas loss-of-function mutations in these genes prevent the ethylene-mediated auxin increase. Furthermore, wei2 and wei7 suppress the high-auxin phenotypes of superroot1 (sur1) and sur2, two auxin-overproducing mutants, suggesting that the roles of WEI2 and WEI7 in the regulation of auxin biosynthesis are not restricted to the ethylene response. Together, these findings reveal that ASA1 and ASB1 are key elements in the regulation of auxin production and an unexpected node of interaction between ethylene responses and auxin biosynthesis in Arabidopsis. This study provides a mechanistic explanation for the root-specific ethylene insensitivity of wei2 and wei7, illustrating how interactions between hormones can be used to achieve response specificity.     

Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators

The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutant ein2-1, and auxin influx mutants aux1-7, aux1-22, and double mutant aux1-7 ein2. Beta-glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains of PS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1 roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nM) restored the root hair length of aux1 mutants to wild-type level, whereas 100 nM NAA was needed for ein2-1 and aux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type and ein2-1 roots, while stimulating these traits in aux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.     

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.     

Sites of auxin action: regulation of geotropism, growth, and ethylene production by inhibitors of auxin transport

The inhibitors of auxin transport-NPA (N-1-naphthylphthalamic acid), DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-a] isoindol-8-one), and TIBA (2,3,5-triiodobenzoic acid)-inhibited geotropism in roots of intact Pisum sativum L. seedlings. NPA and DPX1840 also caused cellular swelling in the roots. The swelling was due to a greater inhibition of elongation than increase in weight and looked identical to the one caused by ethylene. However, ethylene did not act as an intermediate in the action of auxin transport inhibitors because all three failed to stimulate ethylene production and some of their growth-inhibiting effect was retained in the presence of saturating levels of ethylene. In the presence of 10 mum indoleacetic acid the growth-inhibiting effect of auxin transport inhibitors was lost after 18 hours. On the other hand, auxin transport inhibitors did not interfere with the ability of auxin to promote ethylene production. Growth inhibition caused by auxin transport inhibitors was reversible. Pea root sections resumed normal growth following flushing of treated sections with inhibitor-free solutions. Experiments with (14)C-2, 4-dichlorophenoxyacetic acid revealed that the herbicide and auxin transport inhibitors may have the same binding site. It was concluded that a class of structurally dissimilar compounds may share a similar physiological role since they all appear to compete with endogenous auxin for certain binding sites and they all have similar growth-regulating activities.     

Ethylene and vegetative development

The past decade has witnessed a tremendous increase in our understanding of the role of ethylene in plant development. Genes encoding enzymes of the ethylene biosynthesis pathway have been isolated, allowing the manipulation of endogenous ethylene levels in intact plants. In parallel, a collection of ethylene mutants was obtained by using a simple response assay. This resulted in the identification of several genes involved in ethylene signal transduction. The data obtained using these new tools have allowed long-standing hypotheses to be tested, while gaining novel insight into the function of ethylene in development. Recent molecular evidence supported the existence of an intense hormonal cross-talk during plant growth. Numerous processes are controlled by ethylene in a close interaction with auxin, and often it was impossible to differentiate between auxin and ethylene effects. Molecular-genetic tools are now allowing the dissection of these interactions. Ethylene does not seem essential for many developmental processes. Nevertheless, it is of crucial importance for survival in most species as it occupies a key position in the developmental response mechanisms of plants under mechanical stress.     

Mechanisms of brassinosteroids interacting with multiple hormones

Various environmental and internal cues play essential roles in regulating diverse aspects of plant growth and development. Phytohormones usually coordinate multiple stimuli to directly regulate multiple developmental programs. Recent studies have provided progresses into the complexity of their cross talk. Particularly, the signaling pathways of various phytohormones have been revealed, leading to the discovery of the mechanisms of the interplay among different hormone signaling pathways. This review focuses on the recent advances of the signaling cross-talk between brassinosteroids and other hormones, including abscisic acid, auxin, gibberellins, ethylene and jasmonate.Key words: brassinosteroids, plant hormone, abscisic acid, auxin, cross talk, signaling     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic

The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin-insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action.     

The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.Auxin is known to play an important role in plant development (Davies, 1995). However, many aspects of auxin biology remain poorly understood. Auxin is synthesized primarily in young tissues, such as cotyledons, leaves, and roots (Ljung et al., 2001, 2005), and transported to other tissues where it is perceived by members of the TRANSPORT INHIBITOR RESPONSE1 (TIR1) auxin receptor family. Recent studies have dramatically increased our knowledge of auxin transport and signaling (Quint and Gray, 2006; Vieten et al., 2007). However, the pathways of auxin synthesis and their regulation are still relatively unclear.Several indole-3-acetic acid (IAA) biosynthetic pathways have been proposed in plants based on research in plant-associated bacteria (Patten and Glick, 1996; Woodward and Bartel, 2005; Spaepen et al., 2007). There are two major types of pathways: the Trp-dependent and Trp-independent pathways. It has been hypothesized that plants have four Trp-dependent pathways that are generally named after an intermediate. In bacteria, the indole-3-pyruvic acid (IPA) pathway, one of the Trp-dependent pathways, has been described in detail (Koga, 1995; Spaepen et al., 2007). The current model for the IPA pathway involves a Trp aminotransferase oxidatively transaminating Trp to IPA. Subsequently, an IPA decarboxylase converts IPA to indole-3-acetaldehyde, and indole-3-acetaldehyde is oxidized to IAA. The IPA pathway is considered a major IAA biosynthetic pathway in plants, since potential intermediates have been isolated from different species (Sheldrake, 1973; Cooney and Nonhebel, 1991; Koga, 1995; Tam and Normanly, 1998). In addition, Trp transamination activity has been found in many plants (Gamborg, 1965; Forest and Wightman, 1972; Truelsen, 1973). Recently, two groups reported the identification of a gene called TAA1. This gene encodes an aminotransferase that converts Trp to IPA and functions in IAA biosynthesis (Stepanova et al., 2008; Tao et al., 2008).To identify genes that are required for auxin synthesis, transport, and signaling, we previously screened for Arabidopsis (Arabidopsis thaliana) mutants that are resistant to auxin transport inhibitors, such as N-1-napthylpthalamic (NPA; Ruegger et al., 1997). The treatment of seedlings with NPA results in auxin accumulation in the root tip (Ljung et al., 2005). Thus, mutants that are resistant to NPA may have defects in synthesis, transport, or response because roots of these mutants are expected to have lower levels of IAA or reduced sensitivity to IAA. This screen succeeded in isolating mutations in seven genes with weak NPA-resistant phenotypes, including genes related to auxin signaling (TIR1), auxin transport (TIR3), and auxin synthesis (TIR7; Ruegger et al., 1997, 1998; Ljung et al., 2005).Here, we describe the characterization of TIR2, a gene whose function is required for auxin synthesis. Genetic and physiological analyses of the tir2 mutant suggest that TIR2 is required for the Trp-dependent auxin synthesis pathway and functions as a Trp aminotransferase. Molecular cloning of TIR2 reveals that the gene is identical to TAA1 (Stepanova et al., 2008; Tao et al., 2008). We show that auxin regulates expression of TIR2 in a tissue-specific manner. Furthermore, we show that TIR2 is required for temperature-dependent hypocotyl elongation and that high temperature positively regulates expression of the TIR2 gene, suggesting that temperature regulates hypocotyl elongation directly by stimulating auxin synthesis. Finally, we provide evidence that TIR2 functions in a positive regulatory loop required for root gravitropism.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

Ethylene and auxin control the Arabidopsis response to decreased light intensity

Morphological responses of plants to shading have long been studied as a function of light quality, in particular the ratio of red to far red light that affects phytochrome activity. However, changes in light quantity are also expected to be important for the shading response because plants have to adapt to the reduction in overall energy input. Here, we present data on the involvement of auxin and ethylene in the response to low light intensities. Decreased light intensities coincided with increased ethylene production in Arabidopsis rosettes. This response was rapid because the plants reacted within minutes. In addition, ethylene- and auxin-insensitive mutants are impaired in their reaction to shading, which is reflected by a defect in leaf elevation and an aberrant leaf biomass allocation. On the molecular level, several auxin-inducible genes are up-regulated in wild-type Arabidopsis in response to a reduction in light intensity, including the primary auxin response gene IAA3 and a protein with similarity to AUX22 and the 1-aminocyclopropane-1-carboxylic acid synthase genes ACS6, ACS8, and ACS9 that are involved in ethylene biosynthesis. Taken together, the data show that ethylene and auxin signaling are required for the response to low light intensities.